APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1978, p. 473-4790099-2240/78/0036-0473$02.00/0Copyright i 1978 American Society for Microbiology

Vol. 36, No. 3

Printed in U.S.A.

Thermal Resistance of Naturally Occurring Airborne BacterialSpores

J. R. PULEO,1* S. L. BERGSTROM, J. T. PEELER,2 AND G. S. OXBORROWt

Planetary Quarantine Laboratory, Jet Propulsion Laboratory, Eastern Test Range, Cape Canaveral,Florida 32920,' and Division ofMicrobiology, Food and Drug Administration, Cincinnati, Ohio 452262

Received for publication 19 June 1978

Simulation of a heat process used in the terninal dry-heat decontamination ofthe Viking spacecraft is reported. Naturally occurring airborne bacterial spores

were collected on Teflon ribbons in selected spacecraft assembly areas andsubsequently subjected to dry heat. Thermal inactivation experiments were

conducted at 105, 111.7, 120, 125, 130, and 135°C with a moisture level of 1.2 mgof water per liter. Heat,survivors were recovered at temperatures of 135°C whena 30-h heating cycle was employed. Survivors were recovered from all cyclesstudied and randomly selected for identification. The naturally occurring sporepopulation was reduced an average of 2.2 to 4.4 log cycles from 105 to 135°C.Heating cycles of 5 and 15 h at temperature were compared with the standard 30-h cycle at 111.7, 120, and 125°C. No significant differences in inactivation (a0.05) were observed between 111.7 and 120°C. The 30-h cycle differs from the 5-and 15-h cycles at 125°C. Thus, the heating cycle can be reduced if a smallfraction (about 10-' to 10-4) of very resistant spores can be tolerated.

A microbiological profile of the Viking space-craft launched from Cape Canaveral, Florida, in1975, has been reported (18). Since an importantpart of the mission was to search for extraterres-trial life, the spacecraft were subjected to aterminal dry-heat cycle to lessen the probabilitythat terrestrial microorganisms would be trans-ported to the planet Mars. This cycle consistedof a nominal temperature of 111.7 + 1.7°C for aperiod of 23 to 30 h after the coldest contami-nated point reached 111.7°C in an inert environ-ment of nitrogen gas having an oxygen contentof less than 2.5% and a moisture content lessthan 0.097% by weight (25). To verify this heatprocess, the cycle was simulated and evaluatedmicrobiologically under laboratory conditions.Naturally occurring airborne bacterial sporeswere collected on Teflon ribbons (15) and testedfor thermal resistance. Spore populations werecollected in the Manned Spacecraft OperationsBuilding (MSOB) and the Vehicle AssemblyBuilding (VAB), areas used for the assemblyand testing of the Apollo spacecraft (20-22).

In any dry-heat sterilization cycle, there areseveral factors which may influence the effi-ciency of the process, e.g., exposure time, tem-perature, and moisture content. It has beenshown that minor changes in moisture may sig-nificantly affect dry-heat resistance of spores (7,13).

t Present address: Food and Drug Administration, Minne-apolis, MN 55401.

Since previously described studies (7, 13) wereconducted using a pure suspension of Bacillussubtilis subsp. niger, the objective of this studywas to examine the thermal resistance of natu-rally occurring airborne spore populations atseveral moisture levels and temperatures. Theexperimental parameters were chosen to simu-late heat cycles employed for decontaminationof unmanned spacecraft.

MATERIALS AND METMODSAn experimental sterilization facility was estab-

lished at the Planetary Quarantine Laboratory, East-ern Test Range, Cape Canaveral, Fla. The systemconsisted of a temperature-controlled oven (PrecisionScientific Co., model 524, Chicago, Ill.) with a nitrogengas supply of known relative humidity. Water vaporlevels were controlled during the heat cycle, and amoisture analyzer (Beckman Infrared Analyzer model865, Beckman Instruments, Inc., Fullerton, Calif.) wasutilized to monitor the gas flowing over samples in theoven. A temperature programmer was developed andinstalled on the oven, giving flexibility to temperature-time sterilization cycles.The oven was located in a vertical laminar flow

clean bench which was attached to a horizontal lami-nar flow clean bench, and the two benches were en-closed by a Plexiglas canopy and plastic curtains. Thethermal experiments and microbiological assays weredone in a small clean room under laminar flow condi-tions (8, 10).The thermal experiments were conducted in a dry-

heat oven under the following conditions. (i) Volumeof oven, 1.5 cubic feet (ca. 0.043 m3). (ii) Nitrogen flow

473

on May 14, 2020 by guest

http://aem.asm

.org/D

ownloaded from

474 PULEO ET AL.

rate to oven during thermal experimental runs, 0.17m3/h or approximately 3 liters/min. Between runs, theoven was purged with dry nitrogen. (iii) Moisturecontent of nitrogen during thermal runs, 1.2 mg ofwater per liter (1,500 jg/ml) or as indicated. (iv)Thermal profile used, high thermal inertia (Fig. 1).

Sixteen stainless steel tables (17.8 cm wide, 188 cmlong, and 102 cm high), each containing two 7.6- by183-cm (0.139 m2) ribbons of Teflon (5 mil [ca. 0.0254mm], FEP Dupont), intended to simulate spacecraftsurfaces, were used as collection surfaces for naturallyoccurring airborne spores in the MSOB, a class 100,000clean room (19), and in the VAB, representative of anassembly area. The Teflon ribbons were cleaned bywashing in hot tap water plus detergent (Haemo-Sol,Heinecke & Co., Inc.), rinsed three times in hot tapwater, rinsed in distilled water, drained dry, and ster-ilized for 3 h at 175°C. New ribbons were precondi-tioned by two 3-h exposures to dry heat at 175°C.Exposure and assay. Thirty-two sterile ribbons

were exposed for 7 days to intramural air to collectairborne microorganisms in the MSOB or VAB envi-ronments, at which time each ribbon was rolled up,aseptically inserted into a sterile stainless steel holder,and placed into sterile 600-ml beakers covered withaluminum foil.

While handling the ribbons, personnel wore clean-room garments and sterile gloves in the MSOB andsterile gloves in the VAB. Control and test ribbonswere selected randomly, and 8 of the 32 ribbons wereused to determine the initial spore concentration (No).

For each test, a total of 24 test ribbons and 9 sterile

APPL. ENVIRON. MICROBIOL.

control ribbons was used. Eight test ribbons plus threesterile control ribbons were placed on each of threeoven shelves. Ribbons were exposed to dry nitrogenfor 6 to 8 h prior to the start of the heat cycle.

After thermal treatment, each ribbon and stainlesssteel holder was placed into sterile jar. Approximately400 ml of sterile Trypticase soy agar (TSA) broth(broth made according to the formula of TSA minusagar: Trypticase [BBL], 15 g/liter, phytone [BBL], 5g/liter, NaCl, 5 g/liter), supplemented with 0.2%(wt/vol) yeast extract (BBL) and 0.1% (wt/vol) solublestarch (BBL) (15), was added aseptically. The jarswere incubated at 32°C and observed for the presenceof growth at 7 days and weekly for 28 days. The sterilecontrol ribbons were treated in the same manner asthe test ribbons. During assay of ribbons, personnelwore sterile clean-room gowns, masks, hats, andgloves.

Supplemented TSA was used for determining sporecounts on the eight control ribbons. Each controlribbon was rolled up and placed in a sterile jar, and400 ml of sterile buffered rinse solution containing0.02% (vol/vol) solution of Tween 80 (polyoxethylenesorbitan monoleate; Hilltop Research, Inc., Miami-vile, Ohio) was added. The control jars were placed inan ultrasonic bath (tank, LTH60-3; generator, A-300;Branson Instruments, Inc., Stamford, Conn.) contain-ing a 0.3% (vol/vol) solution of Tween 80 for 6 min at25 kHz (16, 17), removed from the tank, manuallyshaken 25 times, and then placed in the ultrasonicbath for an additional 6 min. Spore assays were per-formed by heat shocking 30-ml portions at 80°C for 20

THERMAL PROFILE

TIME/HRS.

FIG. 1. Thermal profile used for determining the thermal resistance of naturally occurring populations.

US1N0a

.4U

IL

on May 14, 2020 by guest

http://aem.asm

.org/D

ownloaded from

HEAT-RESISTANT AIRBORNE BACTERIAL SPORES 475

min before plating. Culture plates were incubated at32°C under aerobic conditions for 7 days, and colonycounts were performed after 2, 3, and 7 days.

Identification of Bacillus spp. surviving heattreatment. Organisms surviving the heat treatmentwere streaked for isolation on supplemented TSA.Cultures were maintained on supplemented TSAslants and transferred to fresh medium 24 h beforeinoculation into test media.

Reduction of nitrate to nitrite was detected withindole-nitrate medium (BBL) by using the assaymethod described by the Society of American Bacte-riologists (23).The Voges-Proskauer test was performed by the

method of Vaughn et al. (26). Tests for the utilizationof citrate, deamination of phenylalanine, decomposi-tion of casein and tyrosine, growth at pH 5.7, andproduction of catalase were determined by methodsdescribed by Gordon et al. (9). Anaerobic growth wasdetermined by a modified version of the method ofGordon et al. (9).

Production of acid from mannitol was observed byusing phenol red broth base (BBL) containing 0.5%(wt/vol) carbohydrate. The ability of the organisms tohydrolyze starch was determined by the technique ofOxborrow and Favero (12). A computerized bacterialidentification system (6) was employed to identify allsurvivors of the heat treatments.

Effect oftemperature. Thermal resistance of nat-urally occurring spore populations was examined atsix temperatures. Spores were collected on Teflonribbons exposed for 7 days in the VAB environmentand subjected to six temperatures (105, 111.7, 120, 125,130, and 135°C) programmed in a dry-heat oven usingthe thermal cycle previously described (Fig. 1). Themoisture content of 1.2 mg of water per liter wasmaintained constant for each of the temperaturesstudied.

Effect of thermal exposure time. Naturally oc-curring spore populations were collected on Teflonribbons as previously described and subjected to threetemperatures (111.7, 120, and 125°C) programmed ina dry-heat oven using the thermal cycle shown in Fig.1. After the heat cycle had reached maximum temper-ature, the time at temperature was varied (5, 15, or 30h), and the moisture content was maintained at 1.2 mgof water per liter.

Effect offallout exposure time. The initial countof naturally occurring airborne spore populations wasobtained from Teflon ribbons which had been exposedto the intramural environment of the VAB for 7 days.A study was done to determine the effect exposuretime might have on the levels of heat-resistant sporescollected after 1 day versus 7 days.

Statistical methods. The counts per Teflon ribbonwere transformed to logarithms to base 10. Replicateobservations were made from initial and final counts,and these counts were used to compute the survivalfraction and log,o reductions. The final spore countwas based on scoring heat-treated Teflon ribbons aspositive or negative for bacterial growth, and theseresults were used to compute a most probable number.Confidence limits (11) were computed on the linearcombination of initial and final counts.Thermal come-up time corrections were computed

as shown by Stumbo (24). Linear regressions wereperformed as demonstrated by Ostle and Mensing(11).

RESULTSOther investigators (4, 13) working with spore

populations have shown a nonlinear relation oflogi0 survivors versus time (constant moistureand temperature). This was anticipated, sincethe individual bacterial components of a mixedpopulation were expected to have differing in-nate resistances to heat. Therefore, the initialcount (No) and the final count, as determined bythe most probable number per Teflon ribbon,were used to calculate the reduction in logio fora given temperature-moisture condition (i.e., 0= logio [initial count] - logio [final count]). Thisresult can also be interpreted as the fraction ofsurvivors, (antilog1o o))'.

Naturally occurring bacterial spores were col-lected from the VAB, and the results from 30-hheat treatments at six temperatures (105, 111.7,120, 125, 130, and 135°C) are shown in Table 1.The initial spore population (No) ranged from230 to 2,900. This population was reduced anaverage of 2.2 log cycles at 105°C to 4.4 logcycles at 135°C. Moisture was controlled at 1.2mg of water per liter during the heat cycle.A number of thernal experiments have been

conducted previously using Teflon ribbonsplaced in the MSOB and VAB. The spore pop-ulation would be expected to change from loca-tion to location and over a given period of time.A typical set of 11 experiments from the MSOByielded a 4 = 3.45593 reduction at 113°C and3.85387 for nine experiments employing a 30-hcycle (113'C) in the VAB. These experimentswere carried out at approximately the same timeand were subjected to a moisture concentrationof 1.2 mg of water per liter. These mixed sporepopulations also indicate different resistance inthe presence of varying moisture conditions. Forexample, the reductions observed in the VABwere 4.43180, 3.85387, and 3.72125 for 0.01, 1.2,and 2.4 mg of water per liter, respectively, at1130C.The standard heating cycle used for the mixed

spore populations in the MSOB and VAB was30 h at temperature (Fig. 1). It has been notedthat most of the Bacillus spp. recovered fromspacecraft are known to have thermal resistanceequal to or less than B. subtilis subsp. niger (5,15). Results from thermal studies have shownthat there is a small fraction (10-3 to 10-4) ofvery resistant spores in the environment. Exper-iments were designed with times of 5, 15, and 30h at three temperatures (111.7, 120, and 125°C)to determine if a reduced heating cycle wouldresult in equivalent reductions. Six experiments

VOL. 36, 1978

on May 14, 2020 by guest

http://aem.asm

.org/D

ownloaded from

TABLE 1. Thermal inactivation of a naturally occurring spore population for six temperaturesa

Temp (°C) Log reduction from Survival fraction Variance' 95% confidence limitsNo105 2.25198 5.6 x 10-3 0.03081

(2)

111.7 2.83014 1.5 x 10-3 0.01354 2.53097-3.12931(5)

120 3.66118 2.2 x 10-4 0.01284 3.36985-3.95251(5)

125 4.33890 4.6 x 10-' 0.00545 4.14910-4.52870(5)

130 4.03724 9.2 x 10-" 0.01216 3.75373-4.32075(5)

135 4.44976 3.6 x 10-i 0.00478 4.27201-4.62751(5)

a At 1.2 mg of water per liter, for a 30-h heating cycle."Parentheses indicate degrees of freedom.

were performed at each of the combinationsexcept for the 5-h cycle at 111.70C. Table 2shows the results from these experiments.

Significant differences were observed in thereduction (4) at 125°C between the 30-h cycleand the 5- and 15-h cycles. However, no differ-ences were noted in the reduction at 1200C for5, 15, and 30 h, or at 111.70C for 15 and 30 h.Analysis of the data obtained at the varioustemperatures demonstrated that a shorter heat-ing cycle will eliminate 99.9 to 99.99% of thespores. Approximately 0.1% of the spores willsurvive 15 h at 111.7°C, and <0.1% will survive5 h at 120 or 1250C.An approximate comparison of resistance can

be achieved by correcting all results for come-up time and reporting results on the same basis.Levels of heat resistance can be expressed interms of the time to effect a 99.99% reduction ininitial population. These values are summarizedin Table 3 and plotted in Fig. 2 for the naturallyoccurring spore population.Experiments were also conducted in the oven

on spores of B. subtilis subsp. niger. Thermalinactivation was estimated for four moistureconditions (0.01, 0.15, 1.20, and 2.40 mg of waterper liter) at 111.70C. The reduction values froman No = 106, using a 1.5-h heating cycle, were4.36942, 2.91196, 1.44634, and 0.84426 for thefour moisture levels. The reduction decreased ina linear manner over this short interval. Thisindicates that an increase in moisture increasesthe resistance of the spore.The resistance of B. subtilis subsp. niger can

be approximately expressed as the time to re-duce the population by 99.99%. The times for

TABLE 2. Thermal inactivation of naturallyoccurring spore populations at three heating cycles

and temperatures (1.2 mg of water per liter)

Time of Reduction0 at temp (00)cycle (h) 111.7 120 125

5 3.65535 3.65212(6) (7)

15 2.77615 3.51134 3.91216(5) (6) (7)

30 2.83014 3.66118 4.33890 b

(6) (6) (6)

aReduction = log10 (initial count) - log10 (finalcount). Parentheses indicate number of experiments.

b Significant at a = 0.05.

TABLE 3. Estimated time in hours to achieve a99.99% reduction in naturally occurring spore

populations from the VAB (1.2 mg of water per liter)Temp (°0) Time (h)

105 67.4111.7 53.6120 41.9125 35.6130 38.5135 35.1

the four moisture conditions were 2.2, 3.3, 6.7,and 11.5 h at 111.70C. The increase in resistancewas more evident in these units. A previousreport (13) in a closed system indicated that the99.99% reduction in population would be 1.5 and3.3 h at 0.01 and 0.2 mg of water per liter,

476 PULEO ET AL. APPL. ENVIRON. MICROBIOL.

on May 14, 2020 by guest

http://aem.asm

.org/D

ownloaded from

HEAT-RESISTANT AIRBORNE BACTERIAL SPORES 477

SO70

50

so

401

a0 -

20-

0I.-o

ae

109a7

4

3

2

- 100 110 120 130

TEMPERATURE °C

FIG. 2. Effects oftemperature on the 99.4tion of naturally occurring bacterial spoitions and a heat-resistant test strain.

respectively, at 1130C. The temperatuby 1.30C but agreement is good. Thussystem used for the mixed naturallyspore populations yielded results similB. subtilis subsp. niger experiments.A total of 212 thermal inactivatio

ments was conducted during this stu5,100 test ribbons were subjected to varmal treatments, and 1,466 showed sigiterial growth after addition of nutriincubation. Of these heat survivors, 73were identified. A listing of these identis shown in Table 4. The Bacillus leatypical Bacillus group constituted thpercentage of organisms recovered.resistant actinomycetes (family Actinceae) represented the next highest grcganisms recovered. These data are cwith those obtained in previous work I

DISCUSSIONThere are many factors to considei

terminal heat cycle can be accepted fspacecraft. The reduction of bacterialan acceptable level is of primary imThis level will be influenced by the 1I

the microorganisms, whether encapsulated, onsurfaces, or in mated surface areas on the space-craft (14). The cycle must also insure the main-tenance of the integrity of the spacecraft com-ponents and systems. Cost and ease of obtainingand maintaining the chosen temperature andrelative humidity must also be considered.

Levels of microbial contamination on surfacesof spacecraft are affected by the type of environ-ment in which the spacecraft is assembled andtested. Naturally occurring spore populationswhich comprise the types of contamination an-ticipated on spacecraft are of unknown compo-sition and depend on the area tested and thelevel of activity in the area. Since the composi-tion of the population is uncontrolled, it seemslikely that chance alone determines the numberand types of resistant spores present. The dataindicate the presence of spores with greater heatresistance than has been previously reported,but to date none have been recovered with theextraordinary heat resistance of Bacillus xero-thermnodurans reported by Bond et al. (1-3),which also was isolated from Cape Canaveralsoil.To assess the geographic distribution of heat-

I resistant microorganisms, Teflon ribbons were140 ISO°exposed to the intramural environments of two

facilities removed from Cape Canaveral. These99% reduc- facilities included an environmentally controlledre popula- area in Denver, Colo., and an area in Pasadena,

Calif., in which no environmental controls wereexercised. After the proper exposure time, the

tres differ ribbons were collected, returned to the Plane->the oven tary Quarantine Laboratory, and subjected tooccurrmng the thermal cycle previously described. Heatlar to the survivors were recovered from these thermal

experiments, and the mean reduction valuesbn experi- were in the same range as those found in the.dy- Over VAB. The diversity of species isolated after ther-ious ther-ns of bac- TABLE 4. Identification of heat survivors fromients and Teflon ribbon studyz iso1atestifications?ntus andte highest[he heat-tomyceta-)up of or-

-onsistent(15).

r before a

,or use on

spores tokportance.)cation of

Organism No. %

B. Ientus 313 42.8B. brevis 35 4.8B. circulans 16 2.2B. subtilis 10 1.4B. coagulans 6 0.8B. 8phaericus 5 0.7B. firmus 4 0.5B. pumilus 3 0.4B. macerans 2 0.3B. licheniformis 1 0.1B. polymyxa 1 0.1Atypical Bacillus 295 40.3Actinomycetes 41 5.6

Total 732 100.0

O NATURALLY OCCURRHJGSPORES - 0.1% RH

0 TEST STRAIN CK4-6- 0.1% RH

VOL. 36, 1978

on May 14, 2020 by guest

http://aem.asm

.org/D

ownloaded from

APPL. ENVIRON. MICROBIOL.

mal exposure was found to be similar regardlessof the geographic origin of the bacterial spores.Some difficulty was encountered in succes-

sively subculturing several of the isolates forinoculation into identification media. This ap-peared to be related to the temperature attainedduring the high thermal cycle. The number ofatypical Bacillus isolated accounted for as muchas 40% of the total isolates. Organisms whichexhibit a lack of biochemical activity are placedinto this category, and may, therefore, not beidentical either morphologically or genetically.Generally, heat survivors were found to have thefollowing characteristics: (i) they producesmaller colonies and grow much slower on lab-oratory media than do non-heat-stressed envi-ronmental isolates of the genus Bacillus; (ii)they sporulate poorly on sporulation media; (iii)they are biochemically less active than non-heat-stressed environmental isolates; and (iv) the ma-jority do not grow anaerobically. Heat-resistantactinomycetes were recovered from tempera-tures up to 1300C.

Fallout exposure time does not appear to af-fect the levels of heat-resistant spores. Airbornespores collected on Teflon ribbons after 1-dayexposure were compared with those collectedafter 7 days. Results showed that the level ofspores collected after 1-day exposure was 103,and this level was increased by less than twofoldafter an additional 6 days. Although the reduc-tions obtained from thermal experiments usingTeflon ribbons exposed for 7 days were higherthan those for 1 day, the difference was notsignificant compared to variation observed inreplicate ribbons.A plot of the inactivation of the naturally

occurring spore population is shown in Fig. 2.These results have been expressed as the timein hours to reduce the initial population 99.99%to allow comparison to a heat-resistant teststrain, CK4-6. This strain was previously iso-lated from an experiment in our laboratory andhas been tentatively identified (A. L. Reyes, A.J. Wehby, R. G. Crawford, J. E. Campbell, andJ. T. Peeler, Abstr. Annu. Meet. Am. Soc. Mi-crobiol. 1977, Q94, p. 277) by ATCC as B. later-osporus. This single strain is less resistant thanthe mixed population, and this indicates thatthere are even more resistant strains in thepopulation. The mixed spore population wasfitted to a linear regression (r = 0.956). Althoughthe linear curve fit was considered adequate todescribe the data, the low value at 125°C mayalso indicate that the resistance is increasing athigher temperatures. This would indicate thatthe mixed populations that survive heatingabove 125°C may be composed of a small frac-

tion of several very resistant organisms that areunaffected by these temperatures.

In determining the initial spore population,fallout ribbons are initially heat shocked at 80°Cfor 20 min to eliminate vegetative cells. Thesame principle can be applied to reduce thenaturally occurring spores and achieve a 99.9 or99.99% reduction by subjecting them to a shortheat treatment. The data show that a heat treat-ment of 5 h at temperature (i.e., 120 and 125°C)will leave a small fraction of very resistantspores. The current study indicated that thetime at temperature must be extended to elimi-nate this resistant fraction. For example, B. xe-rothermodurans must be heated for 139 h at125°C to achieve a 90% reduction in the initialpopulation. Therefore, the time of the heatingcycle can be reduced if a small fraction (about10-3 to 10-') of resistant spores can be tolerated.Tests with B. subtilis subsp. niger demon-

strate and confirm (13) that small changes inmoisture can dramatically affect the time toeliminate spores. However, recent studies(Reyes et al., Abstr. Annu. Meet. Am. Soc. Mi-crobiol. 1977, Q94, p. 277) show that the relationto humidity for each spore may differ. Thus, thedesign of a heating cycle will depend on theresistance profile of the test organism (15). Nat-urally occurring mixed spore populations, al-though the distribution is uncontrolled, do givean approximate picture of heat resistance ofspores and can serve to test a thermal steriliza-tion cycle.

ACKNOWLEDGMENTSThis paper presents the results of one phase of research

carried out at the Jet Propulsion Laboratory, California Insti-tute of Technology, under Contract no. NAS 7-100 sponsoredby the National Aeronautics and Space Administration.We thank J. E. Campbell of the Food and Drug Adminis-

tration for helpful advice and direction during the course ofthis study. We are also indebted to P. D. Stabekis, ExotechResearch and Analysis, Inc., for the helpful discussions andsuggestions and to L. A. Maull for his technical assistance. Wealso thank L. B. Hall, without whose support this work wouldnot have been possible.

IUTERATURE CITED1. Bond, W. W., and M. S. Favero. 1975. Thermal profile

of a Bacillus species (ATCC 27380) extremely resistantto dry heat. Appl. Microbiol. 29:859-860.

2. Bond, W. W., and M. S. Favero. 1977. Bacillus xero-thermodurans sp. nov., a species forming endosporesextremely resistant to dry heat. Int. J. Syst. Bacteriol.27:157-160.

3. Bond, W. W., M. S. Favero, and M. R. Korber. 1973.Bacillus sp. ATCC 27380: a spore with extreme resist-ance to dry heat. Appl. Microbiol. 26:614-616.

4. Bond, W. W., M. S. Favero, N. J. Petersen, and J. H.Marshall. 1970. Dry heat inactivation kinetics of nat-urally occurring spore populations. Appl. Microbiol.20:573-578.

478 PULEO ET AL.

on May 14, 2020 by guest

http://aem.asm

.org/D

ownloaded from

HEAT-RESISTANT AIRBORNE BACTERIAL SPORES 479

5. Bond, W. W., M. S. Favero, N. J. Petersen, and J. H.Marshall. 1971. Relative frequency distribution ofDi25c values for spore isolates from the Mariner-Mars1969 spacecraft. Appl. Microbiol. 21:832-836.

6. Dillon, R. T., D. Holdridge, J. R. Puleo, and G. S.Oxborrow. 1971. A computerized bacterial identifica-tion system as applied to planetary quarantine. SpaceLife Sci. 3:63-84.

7. Drummond, D. W., and L. J. Pflug. 1970. Dry-heatdestruction ofBacillus subtilis spores on surfaces: effectof humidity in an open system. Appl. Microbiol.20:805-809.

8. Favero, M. S., and K. R. Berquist. 1968. Use of laminarair-flow equipment in microbiology. Appl. Microbiol.16:182-183.

9. Gordon, R. E., W. C. Haynes, and C. H.-N. Pang. 1973.The genus Bacillus. Agriculture handbook No. 427.United States Department of Agriculture, U.S. Govern-ment Printing Office, Washington, D.C.

10. National Aeronautics and Space Administration.1967. NASA standards for clean rooms and work sta-tions for the microbially controlled environment. Na-tional Aeronautics and Space Administration, Washing-ton, D.C.

11. Ostle, B., and R. W. Mensing. 1975. Statistics in re-search, 3rd ed. Iowa State University Pres, Ames.

12. Oxborrow, G. S., and M. S. Favero. 1967. A combina-tion medium for demonstrating starch and gelatin hy-drolysis. Am. J. Med. Technol. 33:334-335.

13. Peeler, J. T., A. L Reyes, R. G. Crawford, A. J.Wehby, and J. E. Campbell. 1977. Thermal resistanceof Bacillus subtilis var. niger in a closed system. Appl.Environ. Microbiol. 33:52-58.

14. Pflug, I. J. 1970. Dry heat destruction rates for micro-organisms on open surfaces, in mated surface areas andencapsulated in solids of spacecraft hardware. In Lifesciences and space research VIII. North-Holland Pub-lishing Co., Amsterdam.

15. Puleo, J. R., M. S. Favero, G. S. Oxborrow, and C. M.Herring. 1975. Method for collecting naturally occur-

ring airborne bacterial spores for determining theirthermal resistance. Appl. Microbiol. 30:786-790.

16. Puleo, J. R., M. S. Favero, and N. J. Petersen. 1967.Use of ultrasonic energy in assessing microbial contam-ination on surfaces. Appl. Microbiol. 15:1345-1351.

17. Puleo, J. R., M. S. Favero, and G. J. Tritz. 1967.Feasibility of using ultrasonics for removing viable mi-croorganisms from surfaces. Contam. Control 6:58-67.

18. Puleo, J. R., N. D. Fields, S. L. Bergstrom, G. S.Oxborrow, P. D. Stabekis, and R. C. Koukol. 1977.Microbiological profiles of the Viking spacecraft. Appl.Environ. Microbiol. 33:379-384.

19. Puleo, J. R., N. D. Fields, B. Moore, and R. C. Graves.1970. Microbial contamination associated with theApollo 6 spacecraft during final assembly and testing.Space Life Sci. 2:48-56.

20. Puleo, J. R., G. S. Oxborrow, N. D. Fields, and H. E.Hall. 1970. Quantitative and qualitative microbiologicalprofiles of the Apollo 10 and 1 l spacecraft. Appl. Micro-biol. 20:384-389.

21. Puleo, J. R., G. S. Oxborrow, N. D. Fields, C. M.Herring, and L. S. Smith. 1973. Microbiological pro-files of four Apollo spacecraft. Appl. Microbiol.26:8384845.

22. Puleo, J. R., G. S. Oxborrow, and R. C. Graves. 1969.Microbial contamination detected on the Apollo 9spacecraft, p. 80-83. In Proc. 8th Annu. Technol. Meet.Am. Assoc. Contamination Control, New York. Ameri-can Association for Contamination Control, Boston.

23. Society of American Bacteriologists, Committee onBacteriological Technic. 1951. Manual of methodsfor pure culture study of bacteria. Leaflet V. Routinetests for the descriptive chart, 12th ed.

24. Stumbo, C. R. 1965. Thermobacteriology in food process-ing. Academic Press Inc., New York.

25. Thompson, M. F. 1975. Viking-a decade of AIBS in-volvement. Bioscience 25:705-707.

26. Vaughn, R., N. Mitchell, and M. Levin. 1938. Effect oftemperatures and test reagents on the Voges-Proskauerand methyl red reactions. J. Bacteriol. 36:313-314.

VOL. 36, 1978

on May 14, 2020 by guest

http://aem.asm

.org/D

ownloaded from